We have cloned a cDNA for a novel member of the opioid receptor family, designated as ROR-C, from the rat cerebrum cDNA library using the probe derived from the dopioid receptor subtype cDNA. The deduced amino acid sequence of ROR-C shows high homology with those of ROR-A (rat S-opioid receptor subtype), ROR-B (rat ,&subtype) and ROR-D (rat K-subtype). RNA blot hybridization and in situ hybridization analysis revealed that ROR-C mRNA is expressed in discrete regions of the rat centraf nervous system.

To examine whether the mitogen-activated protein kinase (MAPK) cascade and phospholipase A2 (PLA2) are involved in the signal transduction mechanism of the opioid receptor, the 6-, p-, and K-opioid receptors were stably expressed from cDNA in Chinese hamster ovary cells. Activation of the 6-, p-, and K-receptors by agonists induced a rapid and transient increase in MAPK activity accompanied by reduced electrophoretic mobility of the 42-kDa isoform of MAPK(p42), probably owing to phosphorylation. The opioid receptor-mediated increase in MAPK activity was suppressed not only by pretreatment with genistein, a tyrosine protein kinase inhibitor, but also by prolonged exposure to phorbol 12-myristate 13-acetate and pretreatment with GF 1 09203X, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of PKC as well as tyrosine protein kinase. Furthermore, stimulation of the 6-, p-, and K-receptors with opioid agonists in the presence of A231 87, a calcium ionophore, resulted in an increase in arachidonate release, suggesting that PLA2 is activated by the opioid receptors when the intracellular Ca 2 concentration is elevated. Both MAPK activation and increase in arachidonate release mediated by the opioid receptors were abolished by pretreatment with pertussis toxin, suggesting that these responses are mediated by G 1 or G0 types of GTP-binding regulatory proteins. Key Words: Opioid receptor-cDNA expression-M itogen-activated protein kinase-Phospholipase A2-Chinese hamster ovary cells.

The complete amino acid sequences of rat opioid receptors (designated as ROR-A and ROR-B) have been deduced by cloning and sequencing the cDNAs. The ligand-binding properties of ROR-A and ROR-B expressed from the cloned cDNAs in Chinese hamster ovary cells correspond most closely to those of the pharmacologically defined delta- and mu-opioid receptor subtypes, respectively. RNA blot hybridization analysis revealed that cerebrum and brainstem contain both ROR-A and ROR-B mRNAs, whereas neither ROR-A nor ROR-B mRNAs can be detected in cerebellum.

In genetically occurring non-insulin-dependent diabetes mellitus (NIDDM) model rats (GK rats), the activities of L-and T-type Ca 2 ϩ channels in pancreatic ␤ cells are found to be augmented, by measuring the Ba 2 ϩ currents via these channels using whole-cell patch-clamp technique, while the patterns of the current-voltage curves are indistinguishable.

To elucidate which portions of the opioid receptor molecules are involved in the ligand selectivity, we have expressed chimeric receptors between the rat delta- and mu-opioid receptors from cDNAs and analysed their ligand binding properties. We demonstrate that the major binding determinant for the delta-selective enkephalin-related peptide, [D-Pen2,D-Pen5]enkephalin, resides within the region comprising the transmembrane segments V-VII and the intervening loop regions. On the other hand, the region spanning from the intracellular loop I to the amino-terminal half of the transmembrane segment III is shown to be involved in determining high-affinity binding of the mu-selective enkephalin-related peptides, [D-Ala2, MePhe4,Gly-ol5]enkephalin and [D-Ala2,MePhe4,Met-ol5]enkephalin, whereas the major determinant for binding of the mu-selective alkaloids, morphine and codeine, is demonstrated to exist in the region spanning the transmembrane segments V-VII. These results indicate that distinct regions of the opioid receptor determine the selectivity for the delta- and the mu-selective enkephalin-related peptides and that the binding determinant for the mu-selective alkaloids is distinct from that for the mu-selective enkephalin-related peptides.